ABSTRACT Fracture nonunion poses a significant clinical problem. In the United States, approximately 1.6 million bone fractures encounter prolonged healing or non-union each year. Fracture nonunion treatment usually involves complicated and massive procedures in practice, and sometimes needs multiple surgeries, therefore increases the cost of health care and results in marked patient disability. The major population bearing with these clinical complications are patients with inflammatory conditions, e.g, elder patients, smoking, diabetic or rheumatoid arthritis (RA) patients, highlighting the potential deleterious role of chronic systemic inflammation in fracture r epair. The overarching hypothesis of this proposal is that under inflammatory conditions, Dnmt3b overexpression on stem cells can be achieved in an auto-regulated and feedback-controlled manner and therefore leads to restoration of stem cell differentiation in cell cultures and fracture repair in RA mice. This hypothesis is supported by our preliminary data wherein we show that Dnmt3b is highly expressed in fracture callus during fracture repair and Dnmt3b is the major DNA methyltransferase (Dnmt) responsive to cytokines in MPCs. Relevant to our proposal, we provide evidence that 1) inflammation decreases Dnmt3b expression in MPCs in vivo and in vitro and leads to fracture nonunion; 2) inflammatory signals inhibit Dnmt3b in an NF-?B- dependent manner; and 3) Dnmt3b gain-of-function (GOF) in MPCs shows protective effect from inflammation in vitro and accelerates fracture repair in mice. We therefore propose to further engineer feedback-controlled anti-inflammatory stem cells with Dnmt3b overexpression, making the cells have the capacity to tune inflammation and meanwhile overcome DNA methylation alterations. CRISPR/Cas9 genome editing platform will be used to generate self-regulated anti-inflammatory stem cells and examine its effect against inflammation on stem cell differentiation in vitro and in vivo in the context with fracture repair. Two main Specific Aims are proposed. Specific Aim 1 will establish the effect of Dnmt3b overexpression on restoration of DNA methylation and stem cell differentiation under inflammation in vitro. Specific Aim 2 will establish the protective effect of self-regulated stem cells on fracture nonunion against inflammation in vivo. This work will define Dnmt3b as a novel target to treat fracture nonunion. The customized therapeutic stem cells will open innovative possibilities for more effective treatments to fracture nonunion especially under inflammatory diseases.